Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same

ABSTRACT

Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3&#39; terminus of the DNA sequence encoding the heterologous polypeptides. Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus.

This is a continuation of application Ser. No. 08/284,942 filed Aug. 2,1994, now U.S. Pat. No. 5,578,463, which is a continuation ofapplication Ser. No. 07/413,010 filed Sep. 25, 1989, now U.S. Pat. No.5,364,770, which is a continuation of application Ser. No. 163,219 filedFeb. 26, 1988, which is a continuation of application Ser. No.06/882,224 filed Jul. 7, 1986, now abandoned, which is acontinuation-in-part of application Ser. No. 06/771,374 filed Aug. 29,1985 now abandoned.

FIELD OF THE INVENTION

The present invention is directed to heterologous polypeptides expressedand secreted by filamentous fungi and to vectors and processes forexpressing and secreting such polypeptides. More particularly, theinvention discloses transformation vectors and processes using the samefor expressing and secreting biologically active bovine chymosin andheterologous glucoamylase by a filamentous fungus.

BACKGROUND OF THE INVENTION

The expression of DNA sequences encoding heterologous polypeptides(i.e., polypeptides not normally expressed and secreted by a hostorganism) has advanced to a state of considerable sophistication. Forexample, it has been reported that various DNA sequences encodingpharmacologically desirable polypeptides [e.g., human growth hormone(1), human tissue plasminogen activator (2), various human interferons(6), urokinase (5), Factor VIII (4), and human serum albumin (3)] andindustrially important enzymes [e.g., chymosin (7), alpha amylases (8),and alkaline proteases (9)] have been cloned and expressed in a numberof different expression hosts. Such expression has been achieved bytransforming prokaryotic organisms [e.g., E. coli (10) or B. subtilis(11)] or eukaryotic organisms [e.g., Saccharomyces cerevisiae (7),Kluyveromyces lactis (12) or Chinese Hamster Ovary cells (2)] with DNAsequences encoding the heterologous polypeptide.

Some polypeptides, when expressed in heterologous hosts, do not have thesame level of biological activity as their naturally producedcounterparts when expressed in various host organisms. For example,bovine chymosin has very low biological activity when expressed by E.coli (13) or S. cerevisiae (7). This reduced biological activity in E.coli is not due to the natural inability of E. coli to glycosylate thepolypeptide since chymosin is not normally glycosylated (14). Suchrelative inactivity, both in E. coli and S. cerevisiae, however, appearsto be primarily due to improper folding of the polypeptide chain asevidenced by the partial post expression activation of such expressedpolypeptides by various procedures. In such procedures, expressedchymosin may be sequentially denatured and renatured in a number of waysto increase biological activity: e.g., treatment with urea (13),exposure to denaturing/renaturingpH (13) and denaturation and cleavageof disulfide bonds followed by renaturation and regeneration of covalentsulfur linkages (15). Such denaturation/renaturation procedures,however, are not highly efficient [e.g., 30% or less recovery ofbiological activity for rennin (13)], and add considerable time andexpense in producing a biologically active polypeptide.

Other heterologous polypeptides are preferably expressed in highereukaryotic hosts (e.g., mammalian cells). Such polypeptides are usuallyglycopoly-peptides which require an expression host which can recognizeand glycosylate certain amino acid sequences in the heterologouspolypeptide. Such mammalian tissue culture systems, however, often donot secrete large amounts of heterologous polypeptides when comparedwith microbial systems. Moreover, such systems are technically difficultto maintain and consequently are expensive to operate.

Transformation and expression in a filamentous fungus involvingcomplementation of aroD mutants of N. crassa lacking biosyntheticdehydroquinase has been reported (16). Since then, transformation basedon complementation of glutamate dehydrogenase deficient N. crassamutants has also been developed (17). In each case the dehydroquinase(ga2) and glutamate dehydrogenase (am) genes used for complementationwere derived from N. crassa and therefore involved homologousexpression. Other examples of homologous expression in filamentous fungiinclude the complementation of the auxotrophic markers trpC, (18) andargB (19) in A. nidulans and the transformation of A. nidulans toacetamide or acrylamide utilization by expression of the A. nidulansgene encoding acetamidase (20).

Expression of heterologous polypeptides in filamentous fungi has beenlimited to the transformation and expression of fungal and bacterialpolypeptides. For example, A. nidulans, deficient inorotidine-5'-phosphate decarboxylase, has been transformed with aplasmid containing DNA sequences encoding the pyr4 gene derived from N.crassa (21,32). A. niger has also been transformed to utilize acetamideand acrylamide by expression of the gene encoding acetamidase derivedfrom A. nidulans (22).

Examples of heterologous expression of bacterial polypeptides infilamentous fungi include the expression of a bacterialphosphotransferase in N. crassa (23) Dictyostellium discoideum (24) andCephalosporium acremonium (25).

In each of these examples of homologous and heterologous fungalexpression, the expressed polypeptides were maintained intracellularlyin the filamentous fungi.

Accordingly, an object of the invention herein is to provide for theexpression and secretion of heterologous polypeptides by and fromfilamentous fungi including vectors for transforming such fungi andprocesses for expressing and secreting such heterologous polypeptides.

SUMMARY OF THE INVENTION

The inventor includes novel vectors for expressing and secretingheterologous polypeptides from filamentous fungi. Such vectors are usedin novel processes to express and secrete such heterologouspolypeptides. The vectors used for transforming a filamentous fungus toexpress and secrete a heterologous polypeptide include a DNA sequenceencoding a heterologous polypeptide and a DNA sequence encoding a signalsequence which is functional in a secretory system in a givenfilamentous fungus and which is operably linked to the sequence encodingthe heterologous polypeptide. Such signal sequences may be the signalsequence normally associated with the heterologous polypeptides or maybe derived from other sources.

The vector may also contain DNA sequences encoding a promoter sequencewhich is functionally recognized by the filamentous fungus and which isoperably linked to the DNA sequence encoding the signal sequence.Preferably functional polyadenylation sequences are operably linked tothe 3' terminus of the DNA sequence encoding the heterologouspolypeptides.

Each of the above described vectors are used in novel processes totransform a filamentous fungus wherein the DNA sequences encoding thesignal sequence and heterologous polypeptide are expressed. The thussynthesized polypeptide is thereafter secreted from the filamentousfungus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of the Aspergillus niger glucoamylaseinserts in pGa1 and pGa5.

FIG. 2 depicts the construction of pDJB-gam-1.

FIG. 3 depicts the construction of mp19GAPR.

FIG. 4, 5, 6, and 7 depict the construction of pGRG1, pGRG2, pGRG3, andpGRG4.

FIG. 8 shows the strategy used to generate mp19 GAPR.sup.Δ C1-.sup.Δ C4from mp19 GAPR.

FIG. 9 depicts the construction of pCR160.

FIG. 10 is a partial restriction map of the Mucor miehei carboxylprotease gene including 5' and 3' flanking sequences.

FIGS. 11 A,B, and C is the DNA sequence of Mucor miehei, carboxylprotease including the entire coding sequence and 5' and 3' flankingsequences.

FIG. 12 depicts the construction of pMeJB1 -7.

FIGS. 13 A and B are a partial nucleotide and restriction map of ANS-1.

FIG. 14 depicts the construction of pDJB-3.

FIG. 15 depicts the construction of plasmid pCJ:GRG1 through pCJ:GRG4.

FIG. 16 depicts a restriction endonuclease cleavage map of the 3.7 KbBamHI fragment from pRSH1.

FIG. 17 depicts the construction of pCJ:RSH1 and pCJ:RSH2.

FIG. 18 depicts the expression of H. grisea glucoamylase from A.nidulans.

DETAILED DESCRIPTION

The inventors have demonstrated that heterologous polypeptides fromwidely divergent sources can be expressed and secreted by filamentousfungi. Specifically, bovine chymosin, glucoamylase from Aspergillusniger and Humicola grises and the carboxyl protease from Mucor mieheihave been expressed in and secreted from A. nidulans. In addition,bovine chymosin has been expressed and secreted from A. awamori andTrichoderma reesei. Biologically active chymosin was detected in theculture medium without further treatment. This result was surprising inthat the vectors used to transform A. nidulans were constructed tosecrete prochymosin which requires exposure to an acidic environment(approximately pH 2) to produce biologically active chymosin.

In general, a vector containing DNA sequences encoding functionalpromoter and terminator sequences (including polyadenylation sequences)are operably linked to DNA sequences encoding various signal sequencesand heterologous polypeptides. The thus constructed vectors are used totransform a filamentous fungus. Viable transformants may thereafter beidentified by screening for the expression and secretion of theheterologous polypeptide.

Alternatively, an expressible selection characteristic may be used toisolate transformants by incorporating DNA sequences encoding theselection characteristic into the transformation vector. Examples ofsuch selection characteristics include resistance to variousantibiotics, (e.g., aminoglycosides, benomyl etc.) and sequencesencoding genes which complement an auxotrophic defect (e.g. pyr4complementation of pyr4 deficient A. nidulans, A. awamori or Trichodermareesei or ArgB complementation of ArgB deficient A. nidulans or A.awamori) or sequences encoding genes which confer a nutritional (e.g.,acetamidase) or morphological marker in the expression host.

In the preferred embodiments disclosed a DNA sequence encoding the ANS-1sequence derived from A. nidulans is included in the construction of thetransformation vectors of the present invention. This sequence increasesthe transformation efficiency of the vector. Such sequences, however,are not considered to be absolutely necessary to practice the invention.

In addition, certain DNA sequences derived from the bacterial plasmidpBR325 form part of the disclosed transformation vectors. Thesesequences also are not believed to be necessary for transformingfilamentous fungi. These sequences instead provide for bacterialreplication of the vectors during vector construction. Other plasmidsequences which may also be used during vector construction includepBR322 (ATCC 37017), RX-2 (ATCC 37125), pMB9 (ATCC 37019) and pSC101(ATCC 37032).

The disclosed preferred embodiments are presented by way of example andare not intended to limit the scope of the invention.

Definitions

By "expressing polypeptides" is meant the expression of DNA sequencesencoding the polypeptide.

"Polypeptides" are polymers of α-amino acids which are covalently linkedthrough peptide bonds. Polypeptides include low molecular weightpolymers as well as high molecular weight polymers more commonlyreferred to as proteins. In addition, a polypeptide can be aphosphopolypeptide, glycopolypeptide or metallopoly-peptide. Further,one or more polymer chains may be combined to form a polypeptide.

As used herein a "heterologous polypeptide" is a polypeptide which isnot normally expressed and secreted by the filamentous fungus used toexpress that particular polypeptide. Heterologous polypeptides includepolypeptides derived from prokaryotic sources (e.g., α-amylase fromBacillus species, alkaline protease from Bacillus species, and varioushydrolytic enzymes from Pseudomonas, etc.), polypeptides derived fromeukaryotic sources (e.g., bovine chymosin, human tissue plasminogenactivator, human growth hormone, human interferon, urokinase, humanserum albumin, factor VIII etc.), and polypeptides derived from fungalsources other than the expression host (e.g., glucoamylase from A. nigerand Humicola grisea expressed in A. nidulans, the carboxyl protease fromMucor miehei expressed in A. nidulans, etc.).

Heterologous polypeptides also include hybrid polypeptides whichcomprise a combination of partial or complete polypeptide sequencesderived from at least two different polypeptides each of which may behomologous or heterologous with regard to the fungal expression host.Examples of such hybrid polypeptides include: 1) DNA sequences encodingprochymosin fused to DNA sequences encoding the A. niger glucoamylasesignal and pro sequence alone or in conjunction with various amounts ofamino-terminal mature glucoamylase codons, and 2) DNA sequences encodingfungal glucoamylase or any fungal carboxy protease, human tissueplasminogen activator or human growth hormone fused to DNA sequencesencoding a functional signal sequence alone or in conjunction withvarious amounts of amino-terminal propeptide condons or mature codonsassociated with the functional signal.

Further, the heterologous polypeptides of the present invention alsoinclude: 1) naturally occuring allellic variations that may exist oroccur in the sequence of polypeptides derived from the aboveprokaryotic, eukaryotic and fungal sources as well as those used to formthe above hybrid polypeptides, and 2) engineered variations in the aboveheterologous polypeptides brought about, for example, by way of sitespecific mutagenesis wherein various deletions, insertions orsubstitutions of one or more of the amino acids in the heterologouspolypeptides are produced.

A "biochemically active heterologous polypeptide" is a heterologouspolypeptide which is secreted in active form as evidenced by its abilityto mediate: 1) the biochemical activity mediated by its naturallyoccurring counterpart, or 2) in the case of hybrid polypeptides, thebiochemical activity mediated by at least one of the naturally occurringcounterparts comprising the hybrid polypeptides.

Each of the above defined heterologous polypeptides is encoded by aheterologous DNA sequence which contains a stop signal which isrecognized by the filamentous fungus in which expression and secretionoccurs. When recognized by the host, the stop signal terminatestranslation of the mRA encoding the heterologous polypeptide.

The "filamentous fungi" of the present invention are eukaryoticmicroorganisms and include all filamentous forms of the subdivisionEumycotina (26). These fungi are characterized by a vegatative myceliumcomposed of chitin, cellulose, and other complex polysaccharides. Thefilamentous fungi of the present invention are morphologically,physiologically, and genetically distinct from yeasts. Vegetative growthby filamentous fungi is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such as S.cerevisiae is by budding of a unicellular thallus, and carbon catabolismmay be fermentative. S cerevisiae has a prominent, very stable diploidphase whereas, diploids exist only briefly prior to meiosis infilamentous fungi like Aspergilli and Neurospora. S. cervisiae has 17chromosomes as opposed to 8 and 7 for A. nidulans and N. crassarespectively. Recent illustrations of differences between S. cerevisiaeand filamentous fungi include the inability of S. cerevisiae to processAspergillus and Trichoderma introns and the inability to recognize manytranscriptional regulators of filamentous fungi (27).

Various species of filamentous fungi may be used as expression hostsincluding the following genera: Aspergillus, Trichoderma, Neurospora,Podospora, Endothia Mucor, Cochiobolus and Pyricularia. Specificexpression hosts include A. nidulans (18, 19, 20, 21, 61), A. niger(22), A. awomari, e.g., NRRL 3112, ATCC 22342 (NRRL 3112), ATCC 44733,ATCC 14331 and strain UVK 143f, A. oryzae, e.g., ATCC 11490, N. crassa(16, 17, 23), Trichoderma reesei, e.g. NRRL 15709, ATCC 13631, 56764,56765, 56466, 56767, and Trichoderma viride, e.g., ATCC 32098 and 32086.

As used herein, a "promotor sequence" is a DNA sequence which isrecognized by the particular filamentous fungus for expression purposes.It is operably linked to a DNA sequence encoding the above definedpolypeptides. Such linkage comprises positioning of the promoter withrespect to the initiation codon of the DNA sequence encoding the signalsequence of the disclosed transformation vectors. The promoter sequencecontains transcription and translation control sequences which mediatethe expression of the signal sequence and heterologous polypeptide.Examples include the promoter from A. niger glucoamylase (39,48), theMucor miehei carboxyl protease herein,. and A. niger α-glucosidase (28),Trichoderma reesei cellobiohydrolase I (29) A. nidulans trpC (18) andhigher eukaryotic promoters such as the SV40 early promoter (24).

Likewise a "terminator sequence" is a DNA sequence which is recognizedby the expression host to terminate transcription. It is operably linkedto the 3' end of the DNA encoding the heterologous polypeptide to beexpressed. Examples include the terminator from A. nidulans trpc (18),A. niger glucoamylase (39,48), A. niger α-glucosidase (28), and theMucor miehei carboxyl protease herein, although any fungal terminator islikely to be functional in the present invention.

A "polyadenylation sequence" is a DNA sequence which when transcribed isrecognized by the expression host to add polyadenosine residues totranscribed nRNA. It is operably linked to the 3' end of the DNAencoding the heterologous polypeptide to be expressed. Examples includepolyadenylation sequences from A. nidulans trpc (18), A. nigerglucoamylase (39,48), A. niger α-glucosidase (28), and the Mucor mieheicarboxyl protease herein. Any fungal polyadenylation sequence, however,is likely to be functional in the present invention.

A "signal sequence" is an amino acid sequence which when operably linkedto the amino-terminus of a heterologous polypeptide permits thesecretion of such heterologus polypeptide from the host filamentousfungus. Such signal sequences may be the signal sequence normallyassociated with the heterologous polypeptide (i.e., a native signalsequence) or may be derived from other sources (i.e., a foreign signalsequence). Signal sequences are operably linked to a heterologouspolypeptide either by utilizing a native signal sequence or by joining aDNA sequence encoding a foreign signal sequence to a DNA sequenceencoding the heterologous polypeptide in the proper reading frame topermit translation of the signal sequence and heterologous polypeptide.Signal sequences useful in practicing the present invention includesignals derived from bovine preprochymosin (15), A. niger glucoamylase(39), the Mucor miehei carboxyl protease herein and Trichoderma reeseicellulases (29). However, any signal sequence capable of permittingsecretion of a heterologous polypeptide is contemplated by the presentinvention.

A "propeptide" or "pro sequence" is an amino acid sequence positioned atthe amino terminus of a mature biologically active polypeptide. When sopositioned the resultant polypeptide is called a zymogen. Zymogens,generally, are biologically inactive and can be converted to matureactive polypeptides by catalytic or autocatalytic cleavage of thepropeptide from the zymogen.

In one embodiment of the invention a "transformation vector" is a DNAsequence encoding a heterologous polypeptide and a DNA sequence encodinga heterologous or homologous signal sequence operably linked thereto. Inaddition, a transformation vector may include DNA sequences encodingfunctional promoter and polyadenylation sequences. Each of the abovetransformation vectors may also include sequences encoding anexpressible selection characteristic as well as sequences which increasethe efficiency of fungal transformation.

"Transformation" is a process wherein a transformation vector isintroduced into a filamentous fungus. The methods of transformation ofthe present invention have resulted in the stable integration of all orpart of the transformation vector into the genome of the filamentousfungus. However, transformation resulting in the maintenance of aself-replicating extra-chromosomal transformation vector is alsocontemplated.

General Methods

"Digestion" of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction site. "Partial" digestion refers to incompletedigestion by a restriction enzyme, i.e., conditions are chosen thatresult in cleavage of some but not all of the sites for a givenrestriction endonuclease in a DNA substrate. The various restrictionenzymes used herein are commercially available and their reactionconditions, cofactors and other requirements as established by theenzyme suppliers were used. In general, about 1 microgram of plasmid orDNA fragment is used with about 1 unit of enzyme and about 20microliters of buffer solution. Appropriate buffers and substrateamounts with particular restriction enzymes are specified by themanufacturer. Incubation times of about one hour at 37° C. areordinarily used, but may vary in accordance with the supplier'sinstructions. After incubation, protein is removed by extraction withphenol and chloroform, and the digested nucleic acid is recovered fromthe aqueous fraction by precipitation with ethanol. Digestion with arestriction enzyme may be followed by bacterial alkaline phosphatasehydrolysis of the terminal 5' phosphates to prevent the two ends of aDNA fragment from forming a closed loop that would impede insertion ofanother DNA fragment at the restriction site upon ligation.

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest by a polyacrylamide gelelectrophoresis, identification of the fragment of interest, removal ofthe gel section containing the desired fragment, and separation of theDNA from the gel generally by electroelution (30).

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double-stranded nucleic acid fragments (30). Unless otherwisestated, ligation was accomplished using known buffers in conditions withone unit of T4 DNA ligase ("ligase") per 0.5 microgram of approximatelyequal molar amounts of the DNA fragments to be ligated.

"Oligonucleotides" are short length single or double strandedpolydeoxynucleotides which were chemically synthesized by the method ofCrea et al., (31) and then purified on polyacrylamide gels.

"Transformation" means introducing DNA in to an organism so that the DNAis maintained, either as an extrachromosomal element or chromosomalintegrant. Unless otherwise stated, the method used herein fortransformation of E. coli was the CaCl₂ method (30).

A. nidulans strain G191 (University of Glasgow culture collection) wastransformed by incubating A. nidulans sphaeroplasts with thetransformation vector. The genotype of strain G191 is pabaA1 (requiresp-aminobenzoic acid), fwA1 (a color marker) , mauA2 (monoaminenon-utilizing), and pyrG89 (deficient for orotidine phosphatedecarboxlase). Sphaeroplasts were prepared by the cellophane method ofBallance et al. (21) with the following modifications. To digest A.nidulans cell walls, Novozyme 234 (Novo Industries, Denmark) was firstpartially purified. A 100 to 500 mg sample of Novozyme 234 was dissolvedin 2.5 ml of 0.6M KCl. The 2.5 ml aliquot was loaded into a PD10 column(Pharmacia-Upsulla, Sweden) equilibrated with 0.6M KCl. The enzymes wereeluted with 3.5 ml of the same buffer.

Cellophane discs were incubated in Novozyme 234 (5 mg/ml) for 2 hours,then washed with 0.6M KCl. The digest and washings were combined,filtered through miracloth (Calbiochem-Behring Corp., La Jolla, Calif.),and washed as described (21). Centrifugations were in 50 or 15 mlconical tubes at ca. 1000×g for 10 min. Following incubation on ice for20 min, 2 ml of the polyethylene glycol 4000 solution (250 mg/ml) wasadded, incubated at room temperature for 5 min. followed by the additionof 4 ml of 0.6M KCl, 50 mM CaCl₂. Transformed protoplasts werecentrifuged, resuspended in 0.6M KCl, 50 mM CaCl₂, and plated asdescribed (21). Zero controls comprised protoplasts incubated with 20 μlof 20 mM Tris-HCl, 1 mM EDTA, pH7.4 without plasmid DNA. Positivecontrols comprised transformation with 5 μg of pDJB3 constructed asdescribed herein. Regeneration frequencies were determined by platingdilutions on minimal media supplemented with 5-10 ppm paba and 500 ppmuridine. Regeneration ranged from 0.5 to 5%.

Because of the low transformation frequencies associated with pDJB1, thederivative containing the Mucor acid protease gene (pMeJB1-7) wasexpected to give extremely low transformation frequencies. Consequently,to obtain pmeJB11-7 transformants of A. nidulans, cotransformation wasused. This was accomplished by first constructing a non-selectablevector containing ANS-1, and then transforming sphaeroplasts with amixture of pmeJB1-7 and the non-selectable vector containing the ANS-1fragment. The rationale for this approach was that the ANS-1 bearingvector would integrate in multiple copies and provide regions ofhomology for pMeJB1-7 integration. The ANS-1 vector was prepared bysubcloning the PstI-PvuII fragment of ANS-1 (FIG. 12A and 13B) frompDJB-3 into pUC18 (33).

The two plasmids (pMeJB1-7 and the ANS-1 containing vector) were mixed(2.5 μg each) and the above mentioned transformation protocol followed.

Transformants obtained with vectors PGRG1-pGRG4 and pDJB-gam weretransferred after 3 or 4 days incubation at 37° C. Minimal media agarplates supplemented with 5 ppm p-aminobenzoic acid were centrallyinoculated with mycelial transfers from transformants. Three to fivedays following inoculation of minimal medium plates, spore suspensionswere prepared by vortexing a mycelial fragment in 1 ml distilled H₂ O,0.02% tween-80. Approximately 5×10⁴ spores were inoculated into 250 mlbaffled flasks containing 50 ml of the following medium: (g/l)Maltodextrin M-040 (Grain Processing Corp., Muscatine, Iowa) 50 g, NaNO₃6 g, MgSO₄.7H2O0.5 g, KCl 0.52 g, KH₂ PO₄, 68 g, 1 ml trace elementsolution (34), 1 ml MAZU DF-60P antifoam (Mazer Chemicals, Inc., Gurnee,Ill.), 10 ppm p-aminobenzoic acid, and 50 ppm streptomycin sulfate.Alternatives to MAZU, such as bovine serum albumin or other appropriatesurfactant may be used. Mucor acid protease secretion was tested inAspergillus complete medium (20 g dextrose, 1 g peptone, 20 g maltextract per liter). Carbon source regulation of chymosin secretion byAspergillus nidulans transformants was assessed by measuring secretionin the above-mentioned starch medium relative to the same mediumsupplemented with 1% fructose, sucrose, or dextrose instead of 5%starch. In all cases, the media were incubated at 37° C. on a rotaryshaker (150 rpm). A pDJB3-derived transformant was included as acontrol.

Western blots of the various secreted chymosins and Mucor mieheicarboxyl protease were performed according to Towbin, et. al (35). Dueto the high concentration of salt in chymosin culture broths and theeffect this salt has on gel electrophoresis a desalting step wasnecessary. Pre-poured G-25 columns (Pharmacia, PD10) were equilibriatedwith 50 mM Na₂ HPO₄. pH 6.0. A 2.5 ml aliquot of culture broth wasapplied to the column. The protein was eluted with 3.5 ml of the samebuffer. The heterologous polypeptides present on the blots were detectedby contacting the nitrocellulose blots first with rabbit anti-chymosin(36) or rabbit anti-Mucor miehei carboxy protease serum (36). The blotswere next contacted with goat-anti-rabbit serum conjugated withhorseradish peroxidase (Bio-Rad, Richmond, Calif.) and developed. Priorto loading on the gels, 50 μl of medium (desalted in the case ofchymosin) was mixed with 25 μl of SDS sample buffer. β-mercaptoethanolwas added to a final concetration of 1%. The sample was heated in a 95°C. bath for 5 minutes after which 40-50 μl of sample was loaded on thegel. Each gel was also loaded with 2 μl each of 650, 65 and 6.5 μg/mlchymosin standards and molecular weight markers. Western blots ofpmeDJ1-7 transformants were similarly analyzed except that gelpermeation was not performed.

Protease activity was detected as described by Sokol, et. al. (37).Luria broth was supplemented with 1-1.5% skim milk (Difco) and 30-35 mlwas poured into a 150 mm petri dish. An aliquot of 2 to 5 μl of culturemedium was spotted on the plate. The plate was incubated over night at37° C. in a humidity box. The activity was determined based on theamount of milk clotting occurring on the plate measured in mm. Theplates were co-spotted with dilutions of 100 CHU/ml or 16.6 CHU/mlrennin (CHU-Chr Hansen Unit, Chr Hansen's Laboratorium, A./S.,Copenhagen). The relationship between the diameter of the coagulationzone (mm) and the centration of enzyme is logarithmic.

In order to distinguish between types of proteases, pepstatin, aninhibitor of the chymosin type of carboxyl protease, was used to inhibitprotease activity attributable to chymosin. Samples of chymosin mutantsand control broths were preincubated with a 1:100 dilution of 10 mMpepstatin in DMSO for 5 minutes before analyzing for protease activity.

Glucoamylase secretion by pDJB-gam-1 transformants in 5% starch mediawas assessed using an assay based on the ability of glucoamylase tocatalyze the conversion of p-nitrophenol-a-glucopyranoside (PNPAG) (38)to free glucose and p-nitrophenoxide. The substrate, PNPAG, wasdissolved in DMSO at 150 mg/ml and 3 to 15 μl aliquots were diluted to200 ul with 0.2 M sodium acetate, 1 mM calcium chloride at pH 4.3. A 25μl sample was placed into a microtitre plate well. An equal volume ofstandards ranging from 0 to 10 Sigma A. niger units/ml (Sigma ChemicalCo., St. Louis, Mo.) were placed in separate wells. To each well, 200 μlof PNPAG solution at 2.25 to 11.25 mg/ml was added. The reaction wasallowed to proceed at 60° C. for 0.5 to 1 hour. The time depended uponthe concentration of enzyme. The reaction was terminated by the additionof 50 μl of 2 M trizma base. The plate was read at 405 nm. Theconcentration of enzyme was calculated from a standard curve.

Unless otherwise stated, chromosomal DNA was extracted from filamentousfungi by the following procedure. The filamentous fungus was grown in anappropriate medium broth for 3 to 4 days. Mycelia were harvested byfiltering the culture through fine cheesecloth. The mycelia were rinsedthoroughly in a buffer of 50 mM tris-HCl, pH7.5, 5 mM EDTA. Excessliquid was removed by squeezing the mycelia in the cheesecloth. About 3to 5 grams of wet mycelia were combined with an equivalent amount ofsterile, acid-washed sand in a mortar and pestle. The mixture was groundfor five minutes to form a fine paste. The mixture was ground foranother five minutes after adding 10 ml of 50 mM tris-HCl, pH 7.5, 5 mMEDTA. The slurry was poured into a 50 ml capped centrifuge tube andextracted with 25 ml of phenol-chloroform (equilibrated with an equalvolume of 50 nM tris-HCl, pH 7.5, 5mM EDTA). The phases were separatedby low speed centrifugation. The aqueous phase was saved and reextractedthree times. The aqueous phases were combined (about 20 ml total volume)and mixed with 2 ml of 3 M sodium acetate, pH 5.4 in sterile centrifugetubes. Ice cold isopropanol (25 ml) was added and the tubes were placedat -20° C. for one hour. The tubes were then centrifuged at high speedto pellet the nucleic acids, and the supernatant fluid was discarded.Pellets were allowed to air dry for 30 minutes before resuspending in400 μl of 10 mM tris-KCl, pH 7.5, 1 mM EDTA (TE buffer). Pancreaticribonuclease (Sigma Chemical Co., St. Louis, Mo.) was added to a finalconcentration of 10 μg per ml, and the tubes were incubated for 30minutes at room temperature (30). Ribonuclease was then removed byextraction with phenol-chloroform. The aqueous layer was carefullyremoved and placed in a tube which contained 40 μl of 3M sodium acetate,pH 5.4. Ice cold ethanol was layered into the solution. The DNAprecipitated at the interface and was spooled onto a glass rod. This DNAwas dried and resuspended in a small volume (100 to 200 μl) of TEbuffer. The concentration of DNA was determined spectrophotometricallyat 260 nm (30).

To confirm the chromosomal integration of chymosin DNA sequences inselected transformants Southern hybridizations were performed (30).Spore suspensions of transformants were inoculated into Aspergilluscomplete medium and incubated at 37° C. on a rotary shaker for 24-48hrs. The medium was non-selective in that it was supplemented with 5 ppmp-aminobenzoic acid and contained sufficient uracil for growth of theauxotrophic parent. In effect, these Southerns also tested for thestability of the transformants. The mycelium was filtered, ground insand, and the DNA purified as previously described. Transformant DNA wasthen digested with various restriction enzymes and fragments separatedby agarose gel electrophoresis. Control lanes included digested pDJB3transformant DNA and undigested DNA. Gels were stained with ethidiumbromide, photographed, blotted to nitrocellulose or nytran (Schleicherand Schuell, Keene, N.H.), and probed with radiolabeled plasmids orspecific fragments.

EXAMPLE 1

Expression and Secretion of Aspergillus niger glucoamylase ByAspergillus Nidulans

A. Construction of pGA1

Aspergillus niger (Culture #7, Culture Collection Genencor, Inc., SouthSan Francisco, Calif.) was grown in potato dextrose broth (Difco,Detroit, Mich.) at 30° C. for 3 days with vigorous aeration. ChromosomalDNA was extracted as previously described.

A synthetic oligonucleotide was used as a hybridization probe to detectthe glucoamylase gene from Aspergillus niger. The oligonucleotide was 28bases in length (28 mer) and corresponded to the first 91/3 codons ofthe published glucoamylase coding sequence (39):

    MetSerPheArgSerLeuLeuAlaLeuSer                                                   - 5'ATGTCGTTCCGATCTCTACTCGCCCTGA 3'                                    

The oligonucleotide was synthesized on a Biosearch automated DNAsynthesizer (Biosearch, San Rafael, Calif.) using the reagents andprotocols specified by the manufacturer.

Genomic DNA from Aspergillus niger was analyzed for the presence ofglucoamylase sequences by the method of Southern (30). Briefly, 10 μg ofAspergillus niger DNA was digested with EcoR1 restriction endonuclease.The digested DNA was subjected to electrophoresis on a 1% agarose gelaccording to standard methods (30). DNA was transferred from the gel toa nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, N.H.) byblotting in 10×SSC (1.5 M NaCl, 0.15 M trisodium citrate) (30). DNA wasfixed to the nitrocellulose by baking in an 80° C. vacuum oven, followedby hybridization at low stringency (2,40) with radiolabeledoligonucleotide probe. Radiolabeling of the synthetic oligonucleotidewas done at 37° C. in a 50 μl reaction that contained 70 mM tris-HCl, pH7.5, 10 mm mgCl₂, 5 mM dithiothreitol, 30 pmoles of syntheticoligonucleotide, 20 pmoles of gamma-[32P]ATP (Amersham, Chicago, Ill.;specific activity 5000 Ci/mmol), and 5 units of T4 polynucleotide kinase(New England Biolabs). After hybridization, the filters were washed 15minutes in 2×SSC, 0.1% sodium dodecylsulfate (SDS) and twice in 2×SSC at37° C. Filters were air dried, wrapped in Saran-Wrap (Dow Chemical) andapplied to Kodak XOmat-AR X-ray film at -70° C. to obtain anautoradiographic image. After developing the autoradiogram, a band ofhybridization was clearly visible corresponding to a 3.5 kilobase-pairEcoR1 fragment.

Genomic DNA from Aspergillus niger was digested with EcoR1 andsize-fractionated by polyacrylamide gel electrophoresis according tostandard methods (30). DNA fragments 3 to 4 kb in size were excised andeluted from the gel (30). This DNA fraction was used to generate alibrary of clones in the Escherichia coli cloning vector pBR322 (ATCC37017). The cloning vector was cleaved with EcoR1 and dephosphorylatedwith bacterial alkaline phosphatase (Bethesda Research Labs). A typicaldephosphorylation reaction consisted of 1 μg of digested vector DNA and1 unit of alkaline phosphatase in 50 μl of 50 mM tris-HCl, pH 8.0, 50 mMNaCl. The reaction was incubated at 65° C. for one hour. The phosphatasewas removed by extraction with phenol-chloroform. The EcoR1,size-selected Aspergillus niger DNA was then ligated with EcoR1 cleavedand dephosphorylated pBR322. A typical ligation reaction contained thefollowing: 100 ng each of vector and insert DNAs, 1 unit of T4 DNAligase (Bethesda Research Labs), 25 mM tris-HCl, pH 7.5, 10 mM MgCl₂, 10mM dithiothreitol, and 1 mM ATP in a 10 μl volume. Ligation reactionswere incubated at 16° C. for 18 to 24 hours. The ligated DNA was used totransform competent E. coli. 294 cells (ATCC 31446) prepared by themethod of Morrison (41). Transformants were selected on LB agar plates(30) which contained carbenecillin at a final concentration of 50 μg perml. Transformants which harbored glucoamylase gene sequences wereidentified by colony hybridization methods (30) using theglucoamylase-specific 28 mer as a probe. Hybridizing colonies werepurified, and plasmid DNAs were isolated from each by the alkaline-SDSminiscreen procedure (30). The plasmids selected in this manner allcontained a 3.5 kb EcoR1 fragment which hybridized to the syntheticglucoamylase probe. One such plasmid, designated pGa1, was selected forfurther analysis. A 1.1 kb EcoR1-BglII fragment from the insert in pGa1was subcloned into M13 mp9 (42) and partially sequenced by the dideoxychain termination method (43) to confirm that the cloned DNA encoded theglucoamylase gene. A restriction endonuclease cleavage map of the 3.5 kbEcoR1 fragment contained in pGa1 is depicted in FIG. 1. It was generatedby single and double restriction digests followed by orientation of theDNA fragments with respect to known restriction sites in pBR322 (44).

B. Construction of pGa5

The nucleotide sequence and restriction map of pGa1 indicated that pGalcontained the entire glucoamylase coding region and 221 nucleotides of5' flanking DNA. The sequences in this 5' region were strikingly similarto typical eukaryotic promoter sequences with TATAAAT and CAAT boxeslocated upstream of the ATG start codon (48).

However, to insure that possible upstream activation sites of theAspergillus niger glucoamylase gene were included in the finaltransformation vector a larger genomic fragment which contained at least1000 bp of 5' flanking DNA was cloned. Southern blotting experimentssimilar to those already described identified a 6.5 kb Clal fragmentwhich hybridized to a radiolabeled EcoRI glucoamylase fragment frompGa1. The EcoRI fragment was radiolabeled by nick translation (30) withalpha-[32]PdCTP (Amersham; specific activity 3000 Ci/mmol). A nicktranslation kit (Bethesda Research Labs) was used for the labelingreaction by following the instructions supplied by the manufacturer.Filters were hybridized and washed under stringent conditions (30).

The 6.5 kb ClaI fragment identified by hybridization was cloned in amanner similar to that described previously. Aspergillus niger DNA wasdigested with Clal and size-fractionated by polyacrylamide gelelectrophoresis. DNA fragments migrating between 5.5 and 8 kb wereexcised and eluted from the gel. This fraction was ligated to Clalcleaved and dephosporylated pBR325 (45). The ligation mixture was usedto transform competent E. coli 294 cells. Transformants were selected onLB agar plates containing carbenecillin (50 μg/ml). Colonies whichcontained glucoamylase gene sequences were identified by colonyhybridization (30). Plasmid DNA extracted from hybridizing coloniescontained a 6.5 kb ClaI fragment which included the 3.5 kb EcoR1fragment cloned previously in pGa1. These recombinant plasmids encodedthe Aspergillus niger glucoamylase gene as confirmed by supercoil-DNAsequencing (46) with the synthetic oligonucleotide (28 mer) as asequencing primer. A restriction endonuclease cleavage map of the 6.5 kbClal fragment was constructed using single and double digests of the DNAcloned in pBR325. Restriction sites in the vector DNA were used asreference points to orient the fragment. This restriction map is shownin FIG. 1. Location of the glucoamylase gene was deduced by comparingrestriction sites of pGa5 to those of the previously publishedglucoamylase genes (39, 47, 48). From the mapping data it was estimatedthat approximately 3.3 kb of the 5'-flanking DNA and about 1 kb of3'-flanking DNA were contained within the cloned fragment.

Plasmid pGa5 was deposited with the ATCC on Aug. 28, 1985 in E. coli 294and has been assigned number 53249.

C. Vector for Expression and Secretion of Aspergillus niger Glucoamylase

The 6.5 kb Clal fragment from pGa5 containing the glucoamylase gene wascloned into the E. coli.--Aspergillus nidulans shuttle vector pDJB3 asdepicted in FIG. 2. The pDJB3 shuttle vector possesses a selectablebeta-lactamase gene and origin or replication from E. coli plasmidpBR325, the pyr4 gene from Neuospora crassa which relieves theauxotrophic requirement for uridine in Aspergillus nidulans strain G191,a sequence known as ANS1 from Aspergillus nidulans which promotes a highfrequency of stable integrative transformants in Aspergillus nidulans,unique EcoR1 and Clal restriction sites for cloning. pDJB is constructedas depicted in FIG. 14. Plasmid pFB6 (32) is digested to completion withBglII and partially digested with HindIII. Fragment Bcontaining the pyr4gene (ca. 2 Kb) is purified by gel electrophoresis and ligated intoHindIII/Bam HI digested pBR325 (fragment A) yielding plasmid pDJBp1. TheANS-1 sequence is cloned by ligating EcoRI digested A. nidulans genomicDNA (strain G191 or other FGSC#4- derived strains) into EcoR1 cleavedpFB6. The resulting pool of EcoRI fragments in pFB6 is used to transforma ura3- S. cerevisiae (E.G. ATCC 44769, 44770 etc.). An autonomouslyreplicating plasmid, plntA, is purified from the S. cerevisiaetransformant. pIntA is digested with EcoRI, the ANS-1 fragment ispurified by gel electrophoresis and ligated into EcoRI digested pDJB1,yielding plasmid pDJB2. pDJB2 is partially digested with EcoRI, treatedwith DNA polymerase I (Klenow), and re-ligated to yield plasmid pDJB3.The partial nucleotide sequence and restriction map of the ANS-1fragment is showin in FIGS. 13A and 13B.

Plasmid pGa5 was digested with ClaI and the large fragment (fragment A)was separated from the vector by agarose gel electrophoresis. Thisfragment was ligated with pDJB3 which had been cleaved with ClaI anddephosphorylated (fragment B). The ligation mixture was used totransform competent E. coli 294 cells. Transformants were selected on LBagar supplemented with carbenecillin (50 μg/ml). Analysis of plasmidDNAs from these transformants indicated that the glucoamylase fragmenthad been inserted as expected. Both orientations of the glucoamylasefragment were obtained by screening various transformants. One plasmid,designated pDJB-gam1 was arbitrarily chosen for transformation ofAspergillus nidulans protoplasts.

D. Expression and Secretion of Glucoamylase

Aspergillus nidulans Strain G191 was transformed with pDJB-gam-1 aspreviously described. Five transformants designated pDJB-gam-1-4, 9, 10,11 & 13 were analyzed for glucoamylase activity as previously described.The results are shown in Table I.

                  TABLE I                                                         ______________________________________                                                      Glucoamylase Activity                                             Sample (Sigma Units/ml)                                                     ______________________________________                                        pDJB3         0.129                                                             pDJB-gam-1-4 0.684                                                            pDJB-gam-1-9 0.662                                                            pDJB-gam-1-10 0.131                                                           pDJB-gam-1-11 0.509                                                           pDJB-gam-1-13 0.565                                                           A. niger 2.698                                                              ______________________________________                                    

As can be seen, each pDJB-gam-1 transformant produced more glucoamylaseactivity than the control indicating that biologically activeglucoamylase was expressed and secreted from the transformed fungi.

EXAMPLE 2

Expression and Secretion of Bovine Chymosin from Asperaillus nidulans

Expression vectors were constructed encoding either a natural precursorof bovine chymosin (preprochymosin) or a fusion precursor in which DNAsequences for Aspergillus niger glucoamylase and prochymosin wereprecisely fused. The strategy for the construction of these vectorsinvolved the following steps. First, a DNA sequence containing a portionof the glucoamylase promoter and a portion of the glucoamylase 5'-codingregion was cloned upstream from a DNA sequence corresponding to theamino-terminal portion of preprochymosin. Next, nucleotides between theDNA fragments were deleted by M13 site-specific mutagenesis (40) usingspecific primer sequences. Finally, a segment of DNA containing thefused sequences was incorporated with the remaining portion of theprochymosin sequence into an expression vector which employed the 5'-and 3'- regulatory sequences of the Aspergillus niger glucoamylase gene.These steps are outlined in FIGS. 3 through 7.

A. Construction of mp19 GAPR

Plasmid pGa5 is used to derive a 337 bp EcoR1-RsaI DNA fragment(fragment A) bearing a portion of the glucoamylase promoter and anamino-terminal segment of the coding region. Fragment A was ligated withEcoR1 and Smal digested M13mp19 RF-DNA (fragment B). The ligationmixture was used to transform E. coli. JM101 (ATCC 33876). Clear plaqueswere analyzed for the presence of fragment A by restriction analysis ofthe corresponding RF-DNA. One isolate containing fragment A, designatedmp19R-Rsa was digested with PstI and XbaI and the large fragment(fragment C) was isolated. A small XbaI-PstI sequence (fragment D)derived from pR3 (49) containing 5' preprochymosin sequences; waspurified by electrophoresis and ligated to fragment C to produce thephage template mp19GAPR as shown in FIG. 3.

B. Site Specific Deletion Mutagenesis

As shown in FIG. 8 mp19GAPRΔC1 was derived from mp19 GAPR by deletingthe nucleotides between the glucoamylase signal peptide codons and thecodons for prochymosin by site-specific mutagenesis. Thus, inmp19GAPRΔC1 the glucoamylase signal peptide codons are precisely fusedto the first codon of prochymosin. Site-specific mutagenesis was done aspreviously described (40) except that only one oligonucleotide was usedto prime second strand synthesis on the single-stranded M13 template(FIG. 4) (40). The synthetic oligonucleotide used to derive mp19GAPRΔC1(primer 1) was 5' GCTCGGGGTTGGCAGCTGAGATCACCAG 3'. Plaques containingthe desired deletion were identified by hybridization with the primerradio-labeled as previously described.

In mp19GAPRΔC3 the nucleotides between those immediately preceding theinitiation codon of glucoamylase and the ATG start codon ofpreprochymosin were joined by site-specific nutagenesis using thesynthetic oligonucleotide (primer 3)

    5' ACTCCCCCACCGCAATGAGGTGTCTCGT 3'.                                       

The resulting mutation linked the glucoamylase promoter region preciselyto the initiation codon of preprochymosin as depicted in FIG. 8.

C. Construction of Vectors for the Expression and Secretion of BovineChymosin

As further depicted in FIG. 4 each of the fusions between theglucoamylase sequences and 5' prochymosin sequences (m19GAPRΔC1 andmp19GAPRΔC3) were combined with the 3' prochymosin sequences and theSaccharomyces cerevisiae phosphoglyceratekinase (PGK) terminator in theAspergillus nidulans transformation vector pDJB3. The replicative formof mp19GAPRΔC1 and mp19GAPRΔC3 was digested with EcoR1 and PstI. Thesmaller fragment (fragment 1) was isolated. Plasmid pBR322 was alsodigested with EcoR1 and PstI and the larger vector fragment (fragment 2)was isolated. Fragments 1 and 2 were joined by ligation and used totransform E. coli. 294. A tetracycline resistant colony containingeither plasmid pBR322GAPRΔC1, or pBR322GAPRΔC3 was isolated. Fragment 2was also treated with E. coli. polymerase I (Klenow fragment). Theresulting blunt ended fragment was circularized by ligation and used totransform E. coli 294. One tetracycline resistant colony containingplasmid pBR322ΔRP was isolated and then digested with HindIII and SalI.The larger vector fragment (fragment 3) was isolated. The plasmid pCR160was digested with HindIII and PstI and fragment 5 (containing the yeastPGK terminator fused to 3' prochymosin codons) was isolated. Fragments3, 4, and 5 were joined by ligation and used to transform E. coli 294. Atetracycline resistant transformant containing plasmid pBR322GAPR.sup.ΔC1 or pBR322GAPR.sup.Δ C3 was isolated.

Plasmid pCR160 contains the yeast 2 μm origin of replication to allowits maintenance as a plasmid in yeast, the yeast TRP-1 gene as a yeastselection marker, an E. coli origin or replication and ampicillinresistance gene from the plasmid pBR322, and a prorennin expressionunit. The prorennin expression unit contained the promoter from theyeast PGK gene, the prorennin coding region, and the terminator from thePGK gene. Construction of this plasmid was accomplished as depicted inFIG. 9 in the following manner: Plasmid YEpIPT (50) was partiallydigested with HindIII followed by a complete EcoR1 digestion, and thevector fragment A was isolated. A second plasmid pPGK-1600 (51) waspartially digested with both EcoR1 and HindIII, and the PGK promoterfragment B was isolated. Fragments A and B were ligated to give theintermediate pInt1 which was again partially digested with EcoR1 and theHindIII, and the vector fragment C was isolated. The PGK terminatorfragment D was isolated following HindIII and Sau3A digestion of theplasmid pB1 (52). The prorennin fragment E was isolated by cleaving pR1(49) DNA with EcoR1 and BclI. Fragments C, D, and E were then ligated toproduce the yeast expression plasmid pCR160. The nucleotide sequence ofthe PGK promoter, structural gene and terminator have been reported(53).

Plasmids pBR322GAPRΔC1 and pBR322GAPRΔC3 contain a completetranscriptional unit for each of the forms of prochymosin. Thistranscriptional unit contains a precursor prochymosin coding sequence,the glucoamylase promoter, and the yeast PGK terminator. However,derivatives of these plasmids and plasmids pBR322GAPRΔC2 andpBR322GAPRΔ4, described hereinafter, [designated pint1 (1-4) FIG. 5)]produced no detectable chymosin when used to transform A. nidulans G191.It is not understood why these derivative plasmids failed to express andsecrete chymosin. However, in light of subsequent results it appearsthat the yeast PKG terminator and/or the short glucoamylase promotorsequence in these plasmids is not recognized by A. nidulans G191. Basedon these results, the pBR322GAPRΔC plasmids were further modified.

In the following steps the transcriptional unit was moved onto theAspergillus nidulans transformation vector pDJB3. Additionalglucoamylase 5' flanking sequences were incorporated just 5' of thepromoter to insure the presence of possible upstream activation siteswhich could be involved in regulating expression. Further, the PKGterminator was replaced with the A. niger glucoamylase terminator frompGa5. Specifically, in FIG. 5 each plasmid (pBR322GAPRΔC1 orpBR322GAPRΔC2) was digested with ClaI and fragment 6 was isolated.Plasmid pDJB3 was also digested with Clal and treated with bacterialalkaline phosphatase in order to minimize self-ligation. This digestedplasmid (fragment 7) was joined to fragment 6 and one ampicillinresistant colony containing plasmid pINTI-1 or plntI-3 was isolated.These plasmids were digested with XoI and NsiI and the larger vectorfragment (fragment 8) was isolated. Plasmid pGa5 which contains theentire glucoamylase gene as well as extensive 5' and 3' flankingsequences was digested with XhoI and NsiI and the smaller fragment(fragment 9, containing approximately 1700 bp of these 5' sequences) wasisolated. Fragments 8 and 9 were joined by ligation and used totransform E. coli 294. One ampicillin resistant colony containingplasmid plnt2-1 or pInt2-3 was isolated. These plasmids differ mostsignificantly from the final vectors (see FIG. 7) in that they containthe yeast PGK terminator rather than the glucoamylase terminator.

Additional steps in the construction of chymosin expression vectors areoutlined in FIG. 6. Plasmid pRl (49) was used to isolate a smallBclI-Asp718 DNA fragment (fragment A) which comprised the 3'- end ofprochymosin cDNA. Fragment A was subsequently cloned into pUC18 (33)that was digested with Asp718 and BamHI (fragment B). Similarly, a 1.2kb ClaI-Asp718 DNA fragment (fragment D) was isolated from plasmid pGa5,and cloned into AccI and Asp718 cleaved pUC18 (fragment C). Theresulting intermediate plasmids, pUC-int1 and pUC-int2, were digestedwith SalI and HindIII, and fragments E and F were isolated. Thesefragments were then ligated to produce pUC-int3 which contained the 3'end of prochymosin followed by the glucoamylase terminator sequences ona HindIII-Asp718 fragment (fragment H).

A new cloning vector, designated pBR-link, was created by inserting asynthetic oligonucleotide linker (containing XhoI and ClaI sites) intothe unique BamHI site of pBR322. This linker connoted the followingsequence:

    5' GATCCATCGATCTCGAGATCGATC 3' 3' GTAGCTAGAGCTCTAGCTACCTAG 5'

The larger HindIII-XhoI fragment of this vector (fragment G) waspurified by electrophoresis. Similarly, the XhoI-Asp718 restrictionfragments (fragments I) of plasmids plnt2-1 and plnt2-3 were isolatedelectrophoretically. Fragments G and H were ligated with each of thedifferent I-fragments in a series of three-way ligations to produce theintermediates pint3-1 and plnt3-3. These key intermediates contained theglucoamylase promoter regions, various signal and propeptide fusions tothe prochymosin (or preprochymosin) sequences followed by theglucoamylase terminator region all within convenient ClaI restrictionsites. Because certain Clal sites, such as those in the linker ofpBR-link, are inhibited by E. coli. DNA methylation, the plasmidsplnt3-1 through pInt3-4 were transformed into a dam-strain of E. coli,designated GM48, (ATCC 39099) from which the plasmids were re-isolated.The unmethylated DNA was digested with ClaI and fragment J was purifiedby electrophoresis. Fragment J from each of theglucoamylase-pro-chymosin fusions was subsequently cloned into theunique Clal site of pDJB3 (fragment K) to produce the final expressionvectors pGRG1 and pGRG3.

D. Expression and Secretion of Bovine Chymosin

Aspergillus nidulans G191 was transformed with pGRG1 and pGRG3 aspreviously described.

Five pGRG1 and five pGRG3 transformants were analyzed. Western analysis(not shown) indicated that each transformant secreted a protein whichreacted with anti-chymosin and which migrated at the same or slightlyhigher molecular weight of bovine chymosin. The higher molecular weightspecies may be due to incorrect processing, media effects, orglycosylation. Integration was confirmed for one transformant of pGRG3by Southern hybridization (results not shown). Each transformant wasalso assayed for chymosin activity. The results of this assay are shownin Table II.

                  TABLE II                                                        ______________________________________                                                                  Range of                                               No. of Transformants Chymosin                                                Transformant Tested Activity μg/ml                                       ______________________________________                                        pDJB3       1               0-0.13                                              pGRG1 5   0-1.5                                                               pGRG3 5 0.05-7.0                                                            ______________________________________                                    

These results indicate that pGRG2 and pGRG3 both secrete a protease, atvarious levels, above the pDJB3 control. Occasionally backgroundproteolytic activity was detected in pDJB3 control broths. As will beshown hereinafter this protease activity of transformants is associatedwith the aspartic acid family of carboxyl proteases of which chymosin isa member.

EXAMPLE 3

Expression and Secretion of Fusion Polypeptides from AsperaillusNidulans

Two fusion polypeptides were constructed for expression and secretionfrom A. nidulans. One fusion polypeptide contained an amino-terminalportion consisting of the pro sequence and first ten amino acids ofAsperillus niger glucoamylase and a carboxyl-terminal portion consistingof bovine prochymosin. The second fusion polypeptide contained anamino-terminal portion consisting of the pro sequence only ofAspergillus niger glucoamylase and a carboxyl-terminal portionconsisting of bovine prochymosin.

A. Vectors for Expressing and Secreting Fusion Polypeptides

Vectors encoding the above fusion polypeptides were constructed bydeleting specific sequences from mp19GAPR followed by the samemanipulations as described above for constructing pGRG1 and pGRG3. Asshown in FIG. 8, in mp19GAPRΔC2 the nucleotides between the glucoamylasepropeptide codons and the codons of prochymosin were deleted using thesite-specific mutagenesis method described above. The sequence of theoligonucleotide synthesized for this mutagenesis (primer 2) was

    5' TGATTTCCAAGCGCGCTGAGATCACCAG 3'.                                       

This mutation was intended to fuse the glucoamylase promoter, signalpeptide, and propeptide codons to the first codon of prochymosin. Inmp19GAPRΔC4 the nucleotide sequences between the tenth codon of matureglucoamylase and the codons of prochymosin were deleted by M13site-specific mutagenesis with the synthetic oligonucleotide (primer 4)

    5' TGAGCAACGAAGCGGCTGAGATCACCAG 3'.                                       

This deletion fused the glucoamylase promoter region, signal peptidesequence, propeptide sequence, plus ten codons of the matureglucoamylase to the codons of prochymosin as shown in FIG. 8. Theseexpression and secretion vectors designated as pGRG2 and pGRG4 were usedto transform A. nidulans.

B. Expression and Secretion of Chymosin from Aspergillus NidulansTransformed With PGRG2 and PGRG4

pGRG2 and pGRG4 transformants were cultured as previously described. Theculture medium was assayed for chymosin activity by Western blot andgave results similar to those obtained for pGRG1 and pGRG3. Integrationof one pGRG2 transformant was confirmed by Southern Analysis (resultsnot shown). The results of the chymosin assay are presented in TableIII.

                  TABLE III                                                       ______________________________________                                                                  Range of                                               No. of Transformants Chymosin                                                Transformant Tested Activity μg/ml                                       ______________________________________                                        pDJB3       1                0-0.13                                             pGRG2 1 0.001-0.42                                                            pGRG4 6 0.004-0.75                                                          ______________________________________                                    

Again each of the transformants demonstrated protease activity above thepDJB3 control indicating that a protease was expressed and secreted bythe transformants. As with pGRG1 and pGRG3, these proteases belong tothe aspartic acid family of carboxyl proteases as evidenced by thepepstatin inhibition. Significantly, these results indicate that hybridpolypeptides have been expressed in a filamentous fungus.

EXAMPLE 4

Pepstatin Inhibition Study

Three of the above vectors containing the various constructionsinvolving chymosin were analyzed in the pepstatin inhibition assay asdescribed supra. The results are shown in Table IV.

                  TABLE IV                                                        ______________________________________                                                       Chymosin                                                         Sample Activity (μg/ml)                                                  ______________________________________                                        pDJB3          0                                                                pDJB3 pepstatin 0                                                             pGRG1 0.2                                                                     pGRG1 pepstatin 0.05                                                          pGRG2 0.1                                                                     pGRG2 pepstatin 0                                                             pGRG3 3                                                                       pGRG3 pepstatin 0.6                                                         ______________________________________                                    

The samples preincubated with pepstatin show a marked decrease inactivity indicating that the protease produced by the transformants isof the aspartic acid family of acid proteases to which chymosin is amember. This data together with the results from the Western analysisindicates that biologically active chymosin is expressed and secreted byA. nidulans G191 transformed with pGRG1, pGRG2, pGRG3 and pGRG4.

The variation in the amount of chymosin activity detected for differentvector constructions in Example II and Example III may reflectdifferences in the recognition of the various signals incorporated ineach transformation vector. Within a particular construction, thevariation in chymosin activity may be related to the copy number of thevector incorporated into the fungal genome and/or to the location ofsuch integration.

EXAMPLE 5

Carbon source studies

One vector, pGRG4, was used to transform A. nidulans G191 which wasthereafter grown on the various carbon sources previously described. Theresults of this assay are shown in Table V.

                  TABLE V                                                         ______________________________________                                        Amount of chymosin activity produced on various carbon                          sources (.sup.μ g/ml)                                                              starch glucose     fructose                                                                            sucrose                                    ______________________________________                                        pDJB3     0      0           0     0                                            pGRG4 3.5 3.5 0.9 1.75                                                      ______________________________________                                    

These results clearly show that chymosin is secreted regardless of thecarbon source. This suggests that transcriptional regulation of theglucoamylase promotor is unlike that in A. niger, i.e. not stronglyinducible by starch.

EXAMPLE 6

Expression and Secretion of Mucor meihei Carboxyl Protease

A. Carboxyl Protease Genomic Probe

The partial primary structure of Mucor miehei acid protease (54) wasinspected for the region of lowest genetic redundancy. Residues 187-191(using the pig pepsin numbering system), try-tyr-phe-trp-asp, wereselected. oligonucleotides complementary to the coding sequencecorresponding to this amino acid sequence,

    5'GC(G/A)TCCCA(G/A)AA(G/A)TA(G/A)TA-3',                                   

were synthesized (31) and labelled using gamma 32P-ATP and T4polynucleotide kinase (30).

B. Cloning of Mucor meihei Carboxyl Protease

Genomic DNA from Mucor miehei (Centraal Bureau Voor Schimmelcultures,Holland 370.75) was prepared as follows. Cells grown in YMB medium (3g/l yeast extract, 3 g/l malt extract, 5 g/l peptone, 10 g/l glucose)were collected by centrifugation, washed twice with 0.5M NaCl, andlyophilized. Cell walls were then disrupted by adding sand to the cellsand grinding the mixture with a mortar and pestle. The resulting powderwas suspended (15 ml. per gram dry weight) in a solution containing 25%sucrose, 50 mM Tris-HCl (pH 8.0), and 10 mM EDTA. SDS was added to afinal concentration of 0.1% and the suspension was extracted once with ahalf-volume of phenol and three times with half volumes of chloroform.The final aqueous phase was dialysed extensively against 10 mM Tris-HCl,pH 8.0 and 1 mM EDTA. The DNA was then precipitated by the addition ofsodium acetate, pH 5.5, to a concentration of 0.3 M. followed by theaddition of 2.5 volumes of cold ethanol. Aliquots of this DNA weredigested with a variety of restriction endonucleases according to themanufacturers' directions and then analyzed for sequences complementaryto sequences of the probes described above, using the method ofSouthern. A positively hybridizing band of approximately 2.5 kb(kilobases) was identified in the HindIII digested DNA. HindIII digestedgenomic DNA was separated by polyacrylamide gel electrophoresis and agel fragment containing DNA of 2.0-3.0 kb was electroeluted aspreviously described. The electroeluted DNA, presumed to be enriched forsequences corresponding to the Mucor miehei acid protease gene, wasethanol precipitated. The cloning vector pBR322 (ATCC 37017) wasdigested with HindIII and dephosphorylated using bacterial alkalinephosphatase. In a typical 10 μl reaction 100 ng of vector and 100 mg ofthe size enriched DNA were joined in the presence of ATP and T4 DNAligase. The reaction was used to transform E. coli 294 (ATCC 31446) bythe calcium shock procedure (30). About 2.0×10⁴ ampicillin resistantclones were obtained. Approximately 98% of these contained clonedinserts as indicated by their failure to grown on tetracyclinecontaining medium. These colonies were tested by a standard colonyhybridization procedure for the presence of sequences complementary tothose of the DNA probes. One positively hybridizing colony, containingplasmid pMe5'muc, was found to contain a HindIII insert of the expected2.5 kb size. The termini of this fragment were subcloned into M13sequencing vectors (33) and their sequences determined by the dideoxychain termination method. One terminus contained sequences correspondingto the known amino terminal amino acid sequence of the acid proteasegene. The adjacent 3' region was sequenced in order to obtain more Cterminal coding sequences. The sequencing strategy is shown in FIG. 10.In this way the entire coding sequence for the mature form of theprotein was obtained. The 5' end of the fragment was found to occur 112bp (base pairs) upstream of the codon corresponding the mature aminoterminus. Since this upstream region contained no in frame initiationcodons it was presumed to be part of a propeptide.

In order to obtain DNA containing the initiation codon as well as 5'untranslated sequences a more 5' clone was isolated as follows. AHindIII-ClaI 813 bP 5' subfragment of the pMe5'Cla Hind III insert wasisolated and labelled by the nick translation method (30). This labelledfragment was used to probe ClaI digested Mucor miehei genomic DNA by themethod of Southern. This experiment revealed a single band ofhybridization corresponding to a molecular weight of approximately 1300bp. Size enriched DNA of this size was isolated and cloned into Claldigested and dephosphorylated pBR322 as described above.

Approximately 9000 ampicillin resistant colonies were obtained. About90% of these contained cloned inserts as indicated by their failure togrow on tetracycline containing medium. These colonies were tested by astandard colony hybridization procedure for the presence of sequencescomplementary to those of the nick translated probe. One positivelyhybridizing colony, containing plasmid pMe2, was found to contain a Clalinsert of the expected 1.3 kb size. Sequencing of the ends of thisfragment showed that one terminus corresponded to sequences near theClal site of the Hind III fragment in pMe5'Cla and thus permittedorientation of the fragment which is shown in FIG. 10. Furthersequencing of the ClaI fragment disclosed the initiation codon and 5'untranslated sequences. The entire coding sequence and the 5' and 3'flanking sequences are shown in FIG. 10. Comparison of the deducedprimary structure with that determined by direct amino acid sequencingindicates that the Mucor protein is made as a precursor with anamino-terminal extension of 69 residues. Based on the structuralfeatures generally present in leader peptides it is likely that residues-21 to -1 comprise a leader peptide and that residues 21-69 comprise apropeptide analogous to that found in the zymogen forms of other acidproteases including chymosin and pepsin (55).

C. Yucor meihei carboxyl protease Expression and Secretion Vector

A vector for expressing and secreting Mucor miehei carboxy proteaseincludes the entire native Mucor miehei acid protease transcriptionalunit including the coding sequence, 5' flanking sequences (promoter),and 3' flanking sequences (terminator and polyadenylation site).

The overall strategy for making this vector is depicted in FIG. 12. TheAspergillus nidulans transformation vector pDJB1 was digested with Claland EcoR1 and the larger vector fragment (fragment 1) was isolated. Theplasmid pMe5'Cla was digested with EcoR1 and Clal and fragment 2 wasisolated. This fragment contains the 5' codons of the acid proteasetogether with about 500 bp of 5' flanking sequences. Fragments 1 and 2were joined by ligation and used to transform E. coli 294. Oneampicillin resistant colony containing plasmid pMeJBint was isolated.This plasmid was digested with Clal and treated with bacterial alkalinephosphatase in order to reduce self ligation and is designated fragment3. Plasmid pMe2 was digested with Clal and the smaller fragment(fragment 4) was isolated. This fragment contains the Mucor miehei acidprotease 3' codons and about 1800 bp of 3' flanking sequences. Fragments3 and 4 were joined by ligation and used to transform E. coli 294. Oneampicillin resistant colony containing plasmid pMeJB1-7 was isolated.This vector was used to transform Aspergillus nidulans.

D. Expression and Secretion of Mucor miehei Carboxyl Protease byAspergillus Nidulans

Southern blot analysis of six transformants indicated the presence ofthe entire Mucor miehei acid protease gene in the Aspergillus nidulansgenome (results not shown). In addition, each of the transformants wereanalyzed by Western blots (results not shown) and for acid proteaseactivity. The results of the protease assay are shown in Table VI.

                  TABLE VI                                                        ______________________________________                                                      Protease                                                          Transformant Activity (mg/ml)                                               ______________________________________                                        1             0.003                                                             2 0.007                                                                       3 0.003                                                                       4 0.005                                                                       5 0.005                                                                       6 0.012                                                                     ______________________________________                                    

These experiments demonstrate expression and secretion of a protein thatreacts with specific antibody to Mucor miehei carboxyl protease andwhich has milk clotting activity. The protein has an apparent molecularweight by electrophoretic analysis that is slightly greater than that ofthe authentic (Mucor miehei derived) protein. This may indicate thatAspergillus nidulans glycosylates this glycoprotein to a differentextent than Mucor miehei. Because the Aspergillus nidulans derived Mucormeihei acid protease appears to have the same specific activity as theauthentic material it appears that it has been processed (by the cell orautocatalytically) to the mature form. The unprocessed forms of otheracid proteases such as chymosin and pepsin are zymogens which requireprocessing (autocatalytic) before activity is obtained.

The varying levels of expression in the various transformants mayreflect the position or copy number of the protease gene in theAspergillus nidulans genome. However, the expression and secretion ofbiologically active carboxyl protease indicates the A. nidulansrecognizes the promoter, signal and terminator signals of Mucor mieheicarboxyl protease.

EXAMPLE 7

Expression and Secretion of Chymosin Encoded by pGRG1-4 from A. Awamoriand Trichoderma reesei pyrG Auxotrophic mutants

The plasmids pGRG1 through pGRG4 (pGRG1-4) were also used to transformorotidine-5'-phosphate decarboxylase (OMPCase) deficient mutants of A.awamori and Trichoderma reesei. The pyr4 gene from N. crassa encoded bythe pGRG1-4 plasmid complements these OMPCase mutants in the absence ofuridine to permit the isolation of successful transformants. The thustransformed mutants of A. awamori and T. reesei secreted detectableamounts of bichemically active chymosin into the culture medium.

A. Production of pyrG Auxotrophs

The method used to obtain pyrG auxotrophic mutants of A. awamori and T.reesei involved selection on the pyrimidine analog 5-fluoro-orotic acid(FOA) (56). The mechanism by which FOA kills wild-type cells is unknown.However, in view of the resistance of OMPCase-deficient mutants to FOA,it is likely that the toxicity occurs through conversion of FOA to5-fluoro-UMP. Whether cell death is caused by a flouoridatedribonucleotide or deoxyribonucleotide is uncertain. The followingmethods describe the isolation of OMPCase-deficient (FOA-resistant)mutants of T. reesei and A. awamori:

1. Trichoderma reesei

A fresh spore suspension of T. reesei strain P37 (NRRL 15709) was washedthree times in sterile distilled water containing 0.01% Tween-BO.Fifteen milliliters of this spore suspension (1×10⁷ spores per ml) wereplaced in a sterile petri dish (100×20 mm) with a sterile magneticstirring bar. The lid was removed and the spores were exposed toultraviolet (UV) light at 254 nm (7000 uW per cm2), in the dark at adistance of 25 cm from the UV light source. The spores were stirredconstantly. UW exposure continued for three minutes (sufficient to give70% killed spores). The irradiated spore suspension was collected in a50 ml centrifuge tube and stored in the dark for one hour to preventphotoreactivation. Spores were pelleted by centrifugation and the pelletwas resuspended in 200 ul of sterile water containing 0.01% Tween-80.

The suspension was diluted and plated onto YNB agar medium (0.7% yeastnitrogen base without amino acids, 2% glucose, 10 mm uridine, 2% agar)(56) containing 0.15% FOA (SCM Specialty Chemicals, Gainsville, Fla.).After 4 days incubation at 30° C., 75 colonies were picked to fresh YNBagar that contained FOA. Sixty-two of the 75 colonies grew and weretoothpicked to minimal agar (6 g/l NaKO₃, 0.52 g/l RCl, 1.52 g/l KH₂PO₄, 1 ml/l trace elements solution, 1% glucose, 0.1% MgSO₄, 20 g/lagar) and minimal agar plus 1 mg/ml uridine to determine uridinerequirements. All of the 62 isolates grew on minimal agar with uridine,but 9 isolates failed to grow on minimal agar alone. These 9 strainswere repicked to minimal agar and minimal agar with uridine. Two of thestrains grew only on minimal agar supplemented with uridine. One ofthese, designated T. reesei pyrG29, grew well on minimal medium withuridine with no background growth on minimal medium alone.

2. Aspergillus awamori

(i) Production of A. awamori strain UVK 143f- a Hyperproducer ofGlucoamylase

Spores of A. awamori strain NRRL 3112 were obtained after 5-7 daysgrowth on Potato Dextrose Agar (PDA, Difoo Co.) at 30° C. Spores wereharvested by washing the surface of the plate with sterile 0.1% Tween-80in distilled H₂ O and gently scraping the spores free. Spores werewashed by centrifugation and resuspended in the same buffer to give afinal concentration of between 1×10⁷ to 2×10⁸ spores/ml. Preparationswere stored at 4° C.

Two ml of spores was added to a sterile petri plate. The top of the dishwas removed and spores were exposed to an ultraviolet (UV) lamp (15watt, germicidal). Conditions of time exposure and distance from thelamp were adjusted such that 90 to 99.9% of the spores were killed.Surviving spores were plated onto PDA medium and grown to form discreteindependent colonies.

Spores from individual mutagenized colonies were inoculated into 50 mlof screening media consisting of 5% corn meal, 0.5% yeast extract, 2%corn steep liquor, adjusted to pH 4.5 prior to sterilization in 250 mlflasks. However, any number of media containing corn or corn starch asthe carbon source would be expected to give similar results. Cultureswere grown for 4-5 days at 30-35° C. with constant shaking. Samples wereremoved either daily or at the end of the run for assays.

Estimates of glucoamylase activity were made by measuring the release ofa color producing group (para-nitro-phenol) from a colorless substrate(para-nitro-phenyl-alpha-glucoside, PNPAG).

The following protocol was utilized:

Substrate--180 mg PNPAG was dissolved in 250 ml of 0.1M NaAcetatebuffer, pH 4.7. Store at 4° C.

Assay--1 ml of substrate was equilibrated at 40° C. in a water bath. 0.2ml of sample (or diluted sample) was added and incubated for 30 minutesat 40° C. 9 ml of 0.1M Na₂ CO₃ was added with the mixture being kept atroom temperature for 15 minutes for color development. The mixture wasfiltered through Wattman 42 filter paper and the absorbance at 420 nmwas read in a suitable spectrophotometer. All mutant PNPAG levels werecompared to the standard amount produced by the parent strain and werereported as percent of PNPAG hyrolysis of the parent.

One glucoamylase-hyperproducing strain designated UVK 143f was selectedfor auxotrophic mutagenesis.

(ii) Auxotrophic Mutagenesis

Preparation of spores from A. awamori strain UVK143f, UV mutagenesis,and mutant analysis were the same as for T. reesei with the followingmodifications:

a. 2.5 minutes was required to give 70% killing with UV light.

b. Minimal medium was used instead of YNB agar.

C. The FOA concentration was 0.1%.

Fifteen pyrG mutants were found. Three of these isolates, designatedpyr4-5, pyr4-7, and pyr4-8 were selected for transformation experiments.

B. Transformation of A. awamori and T. reesei pyr Auxotrophs

A. awatnori and T. reesei auxotrophs were transformed by a modificationof the procedure previously described for A. nidulans. Approximately1×10⁸ spores were inoculated into yeast extract glucose (YEG) mediumwhich is 2% glucose, 0.5% yeast extract supplemented with 1 mg/mluridine. The cultures were incubated on a 37° C. shaker (200 rpm) for 12to 15 hours [T. reesei was incubated at 30° C.]. Germlings wereharvested by centrifugation, washed once with sterile YEG medium, thenincubated at 30° C. in 50% YEG medium containing 0.6 M KCl, 0.5%Novozyme 234 (Novo Industries, Denmark), 0.5% MgSO₄.7H₂ O, 0.05% bovineserum albumin in a sterile 200 ml plastic bottle (Corning Corp.,Corning, N.Y.). After 30 minutes of shaking at 150 rpm, the protoplastsuspension was vigorously pipetted up and down five times with a 10 mlpipette to disperse the clumps. The protoplast suspension was furtherincubated as above for one hour then filtered through sterile miracloth(Calbiochem-Behring Corp., LaJolla, Calif.) that was wet with 0.6 M KCl.The filtered protoplasts were centrifuged, washed, and transformed witheach of the plasmids pGRG1-4 as described previously.

The following modifications were made for A. awamori transformation:

1. 0.7 M KCl was used instead of 0.6 M KCl.

2. 1.2 M sorbitol was used instead of 0.6 M KCl to osmotically stabilizethe transformation and regeneration medium.

C. Analysis of A. awamori and T. reesei Transformants

Both A. awamori and T. reesei transformants secreted chymosinpolypeptides into the culture medium. This was determined by analyzingculture filtrates (results not shown) for both enzymatically activechymosin (milk clotting assay) and chymosin polypeptides that reactedwith specific chymosin antibodies (enzyme immunoassays and Westernimmunoblotting techniques).

EXAMPLE 8

Expression and Secretion of Heterologous Polypeptides from argBAuxotrophic Mutants of Aspergillus Species

The expression and secretion of heterologous polypeptides from argBauxotrophs of Aspergillus species has also been achieved.

This example describes the complementary transformation of A. nidulansand A. awamori argB auxotrophs with vectors containing the ArgB genefrom A. nidulans and DNA sequences encoding the heterologouspolypeptides of plasmids pGRG1-4. The argB gene encodes ornithinetranscarbamylase (OTC).

The A. nidulans argB auxotroph containing the genetic markers biA1,argB2, metG1 used herein was obtained from Dr. P. Weglenski, Departmentof Genetics, Warsaw University, Al. Ujazdowskie 4,00-478 Warsaw, Poland.The A. awamori argB mutant was derived as follows.

A. Isolation of Aspergillus awamori argB Auxotrophic Mutants

A fresh suspension of A. awamori strain UVK 143k spores was prepared andUV mutagenesis was performed as described above except that the exposuretime was sufficient to kill 95% of the spores. The spores were thencentrifuged, washed with sterile water, and resuspended in 25 ml ofsterile minimal medium. These suspensions were incubated in a 37° C.shaker with vigorous aeration. Under these conditions, wild-type sporeswill germinate and grow into vegetative mycelia, but auxotrophic mutantswill not. The culture was aseptically filtered through sterile miraclothevery six to eight hours for three days. This step removes most of thewild-type mycelia while the ungerminated auxotrophs pass through themiracloth filter (i.e., filtration enrichment). At each filtration stepthe filtered spores were centrifuged and resuspended in fresh minimalmedium. After three days of enrichment the spores were diluted andplated on minimal agar supplemented with 50 mM citrulline. The plateswere incubated at 37° C. for two to three days. Individual colonies weretoothpicked from these plates to two screening plates--one plate thatcontained minimal agar plus 10 mM orhithine and one plate that containedminimal agar plus 50 mM citrulline. The rationale for picking coloniesto these screening plates is as follows. OTC (the argB gene product)catalyzes the conversion of ornithine to citrulline in the argininebiosynthetic pathway. Thus argB mutants (deficient in OTC) will grow onminimal medium plus citrulline but not on minimal medium with ornithine.Screening of approximately 4000 colonies by this method yielded 15possible argB mutants. One of these strains, designated A. awamoriargB3, gave no background growth on minimal medium and grew very well onminimal medium supplemented with either arginine or citrulline. Assaysto determine the level of OTC activity (57) indicated that the argB3mutant produced at least 30-fold less OTC activity than wild-type. Onthe basis of these data the A. awamori argB3 strain was selected fortransformation experiments.

B. Construction of argB-based Prochymosin Expression Vectors forTransformation of Aspergillus Species

In this construction (see FIG. 15) the first step was to combine thetransformation enhancing sequence ANS-1 and the selectable argB gene onthe same plasmid. In order to achieve this, plasmid pBB116 (59), whichcontains the argB gene from A. nidulans, was digested with PstI andBamHI and the indicated fragment A, which contains the argB structuralgene, was isolated. Plasmid pDJB2 (59) was digested with EcoR1 and PstI,and the indicated fragment B, which contains the ANS-1 sequence, wasisolated. In a three part ligation fragments A and B were joinedtogether with fragment C, which contains the large EcoR1-BamHI fragmentof plasmid vector pUC18 (33) to give plasmid pARG-DJB.

In the second step of this construction a synthetic DNA polylinkercontaining ClaI sites was inserted into pARG-DJB in order to allow theinsertion of Clal fragments which contain various prochymosin expressionunits. Plasmid pARG-DJB was digested with BamHI and thendephoshporylated with bacterial alkaline phosphatase. The indicatedsynthetic DNA polylinker was phosphorylated with T4 polynucleotidekinase, and then ligated to the cleaved PARG-DJB to give pCJ16L. Becausethis plasmid was found to be resistant to digestion with Clal, it wasfirst used to transform the E. coli dam- mutant strain GM48 in order toprevent methylation of the Clal sites. Upon isolation of the plasmidfrom GM48 transformants, it was successfully cleaved with Clal anddephosphorylated with bacterial alkaline phosphatase.

In the final step of this construction of ClaI-cleaved pCJ16L vector wascoined to each of the ClaI prochymosin expression fragments fromplasmids pGRG1 through pGRG4. The resulting four plasmids, pCJ::GRG1through pCJ::GRG4, were used to transform the argB mutants of A.nidulans and A. awamori to prototrophy. Resulting transformants wereanalyzed for expression of prochymosin polypeptides.

C. Analysis of A. nidulans and A. awamori transformants

Secreted chymosin polypeptides from A. awamori and A. nidulanstransformed with pCJ::GRG1 through pCJ::GRG4 were detected by the milkclotting assay and by enzyme immunoassays and Western immunoblottingtechniques. In each case (results not shown) the transformed fungisecreted biochemically active chymosin into the culture medium.

EXAMPLE 9

Expression and Secretion of Humicola grisea glucoamylase from A.nidulans

The glucoamylase gene from the fungus Humicola grisea was isolated andcloned. This gene was thereafter ligated into the argB expressionplasmid pCJ16L. The resulting vectors, pCJ:RSH1 and pCJ:RSH2 were usedto transform argB deficient A. nidulans (Example 8) which resulted inthe expression and secretion of Humicola grisea glucoamylase.

A. Isolation and Cloning of Humicola grisea Glucoamylase Gene

1. Purification of Humicola grisea Glucoamylase

Authentic H. grisea (var. thermoidea NIRL 15219) glucoamylase wasobtained from A. E. Staley Company (lot no. 1500-149-8A). The enzyme waspurified to homogeneity through chromatography on a 4.6 mm×250 mmSynchrompak C4 reversed phase column (Synchrom, Inc., Linden, Ind.). Thecolumn was initially equilibrated with 0.05% triethylamine and 0.05%trifluoroacetic acid (solvent A) at 0.5 ml/min. After injection of theglycomaylase sample (40 μg) the column was washed for 2 minutes withsolvent A, and then eluted with a gradient of 5% solvent B per minute(solvent B is 0.05% triethylamine, 0.05% trifluoroacetic acid inacetonitrile) to 40% solvent B. The slope of the gradient was thenchanged to 0.5% solvent B per minute, and the glucoamylase was eluted atapproximately 55% solvent B. At this point the glucoamylase was judgedto be homogenous by sodium dodecylsulfate polyacrylamide gelelectrophoresis.

2. Amino Acid Sequence of H. grisea Glucoamylase

The amino terminal sequence of purified H. grisea glucoamylase wasobtained as described previously (60). The sequence read as follows:

    AAVDTFINTEKPSAXNSL

These and other lettered peptide sequences presented herein refer toamino acid sequences wherein each letter corresponds to the followingamino acids:

    ______________________________________                                        Amino acid                                                                      or residue 3-letter 1-letter                                                  thereof symbol symbol                                                       ______________________________________                                        Alanine          Ala         A                                                  Glutamate Glu E                                                               Glutamine Glu Q                                                               Aspartate Asp D                                                               Asparagine Asn N                                                              Leucine Leu L                                                                 Glycine Gly G                                                                 Lysine Lys K                                                                  Serine Ser S                                                                  Valine Val V                                                                  Arginine Arg R                                                                Threonine Thr T                                                               Proline Pro P                                                                 Isoleucine Ile I                                                              Methionine Met M                                                              Phenylalanine Phe F                                                           Tyrosine Tyr Y                                                                Cysteine Cys C                                                                Tryptophan Trp W                                                              Histidine His H                                                             ______________________________________                                    

In order to obtain peptide fragments for additional amino acidsequencing, purified glucoamylase (1 mg/ml) was digested in 2% aceticacid for 2 hours at 108° C. The material was injected directly onto aSynchrompak C4 column (4.8 mm×100 mm) equilibrated as described above.After washing for 2 minutes with 100% solvent A (see above), thepeptides were eluted with a linear gradient of solvent C (1% perminute). Solvent C was composed of 0.05% triethylamine, 0.05%trifluoroacetic acid in propanol. At this point three peptides wereselected for further analysis. One peptide (GA3) was sequenced directly.A mixture of two other peptides (GA1 and GA2) was subjected to furtherpurification on a 4.8 mm×250 mm Synchrompak C4 column as follows. Themixture of GA1 and GA2 was diluted with three volumes of solvent A andinjected onto the column. After washing for 2 minutes, the peptides wereeluted with a linear gradient of solvent D (0.5% per minute). Solvent Dwas 0.05% triethylamine, 0.05% trifluoroacetic acid in 35% propanol:65%acetonitrile. Separated GA1 and GA2 were then purified again using thesame protocol and the amino acid sequences were determined as describedabove. The sequences of peptides GA1, GA2 and GA3 are as follows:

    GA1   PLWSITVPIKATGXAVQYKYIKVXQL                                                 - GA2 AAVRPLINPEKPIAWNXLKANIGPN                                               - GA3 INTEKPIAWNKLLANIGPNGKAAPGAAAGVVIASPSRTD                          

3. Synthetic Oligonucleotide Probes

The genomic DNA encoding the H. grisea glucoamylase gene was cloned asfollows. A synthetic mixture of 48 oligonucleotides was used as ahybridization probe to detect the glucoamylase gene. Theoligonucleotides were 17 bases in length (17 mer) and corresponded to asequence of six amino acids (underlined in the GA1 peptide, supra) fromH. grisea glucoamylase:

              Gln Tyr Lys Tyr Ile Lys                                               5' CAA TAT AAA TAT ATT AA 3'                                                       G   C   G   C   C                                                                             A                                                  

The mixture of 48 oligonucleotides was synthesized in six pools, eachcontaining eight different synthetic 17 mers.

    pool 1:        5' CAATATAAATATATTAA 3'                                                G  C  G                                                                  - pool 2: 5' CAATATAAATACATTAA 3'                                                  G  C  G                                                                  - pool 3: 5' CAATATAAATATATCAA 3'                                                  G  C  G                                                                  - pool 4: 5' CAATATAAATACATCAA 3'                                                  G  C  G                                                                  - pool 5: 5' CAATATAAATATATAAA 3'                                                  G  C  G                                                                  - pool 6: 5' CAATATAAATACATAAA 3'                                                  G  C  G                                                           

The oligonucleotides were synthesized on a Biosearch automated DNAsynthesizer (Biosearch, San Rafael, Calif.) using reagents and protocolsspecified by the manufacturer.

4. Selection of Correct Oligonucleotide Probe

Genomic DNA from H. grisea was analyzed for the presence of glucoamylasesequences by the method of Southern (30). Briefly, H. grisea DNA wasdigested with BamHI restriction endonuclease. Six aliquots of thisdigested DNA (one for each probe pool) were fractionated according tosize by electrophoresis on a 1% agarose gel. After blotting the DNA tonitrocellulose, as previously described, the DNA was fixed to thenitrocellulose filter at 80° C. in a vacuum oven. The filter was cutinto six strips, corresponding to the six aliquots of BamHI digestedDNA, and each strip was hybridized for 18 hours at low stringency (2,40) with one of the pools of synthetic oliconucleotide probes. (Theprobes were radiolabeled with gamma-[32P]ATP using T4 polynucleotidekinase as previously described.) After hybridization, the filters werewashed 15 minutes in 2×SSC, 0.1% SDS at 37° C., and twice in 2×SSC atthe same temperature. Filters were air dried, wrapped in Saran-Wrap, andapplied to Kodak XOmat-AR X-ray film to obtain an autoradiographicimage. After developing the autoradiogram, a faint band of hybridizationcorresponding to a 3.7 Kb BamHI fragment was visible from the strip thatwas hybridized with pool 3.

In order to improve the hybridization signal, pool 3 was re-synthesizedas eight individual oligonucleotides. The Southern hybridizationexperiments were repeated using each of the eight oligonucleotides asprobes. Only one of these 17 mer probes was found to hybridize to the3.7 Kb BamHI fragment of H. grisea genomic DNA. The sequence of theoligonucleotide was 5' CAGTACAAGTATATCAA 3'. This 17 mer was used as thehybridization probe for the cloning of the H. grisea glucoamylase gene.

5. Cloning of Glucoamylase Gene Sequences

Genomic DNA from H. grisea was digested with BamHI and size-fractionatedby polyacrylamide gel electrophoresis according to standard methods(30). DNA fragments 2 to 4 Kb in size were excised and eluted from thegel. This DNA fraction was used to generate a library of clones in theE. coli cloning vector pBR322 (ATCC 37017). The cloning vector wasdigested with BamHI and dephosphorylated with bacterial alkalinephosphatase. The phosphatase was removed by extraction withphenol-chloroform (1:1 v/v). The BamHI cleaved size-selected H. griseaDNA was ligated to the BamHI cleaved and dephosphorylated pBR322. Thethus ligated DNA was used to transform competent E. coli 294 cells (ATCC31446) prepared by the method of Morrison (41). Transformants wereselected on LB agar plates (30) which contained carbenecillin at aconcentration of 50 μg/ml. Transformants which harbored glucoamylasegene sequences were identified by colony hybridization methods (30)using the specific 17 mer (described above) as a probe. Hybridizingcolonies were purified, and plasmids were isolated from each by thealkaline-SDS miniscreen procedure (30). The plasmids selected in thismanner all contained a 3.7 Kb BamHI fragment which hybridized to theglucoamylase-specific 17 mer probe. One such plasmid, designated pRSH1,was selected for further analysis.

A 600 bp Sau3A fragment from pRSH1 was subcloned into bacteriophageM13mp18 (33) and partially sequenced by the dideoxy chain terminationmethod (43) to confirm that the cloned DNA encoded the glucoamylasegene. A restriction endonuclease cleavage map of the 3.7 Kb BamHIfragment contained in pRSH1 is shown in FIG. 16. It was generatedfollowing single and double restriction digests followed by orientationof the DNA fragments with respect to known restriction sites in pBR322(44). On the basis of the DNA sequencing data we obtained and therestriction map, we determined that there was a high probability thatthe entire coding sequence of the glucoamylase gene was contained withinthe 3.7 Kb BamHI fragment in pRSH1.

B. Construction of argB Vector Containing Humicola grisea GlucoamylaseGene

The 3.7 Kb BamHI fragment from pRSH1 was cloned (in both orientations)into pCJ16L which contains a selectable argB gene from A. nidulans (FIG.17). The resulting vectors, PCJ:RSH1 and pCJ:RSH2, were used totransform argB-deficient A. nidulans.

C. Expression and Secretion of H. grisea alucoamylase

Prototrophic transformants were purified and innoculated into minimalmedium with starch as the sole carbon source (this medium is the same asthat described for the production of chymosin except that the pH wasadjusted to 5.0). Culture filtrates were assayed for H. griseaglucoamylase activity. FIG. 18 shows the extracellular production of H.grisea glucoamylase by A. nidulans transformed with pCJ:RSH1. Thenegative control was non-transformed ArgB deficient A. nidulans.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those ordinarily skilled in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention.

The references grouped in the following bibliography and respectivelycited parenthetically by number in the foregoing text, are herebyincorporated by reference.

BIBLIOGRAPHY

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What is claimed is:
 1. A process comprising transforming a filamentousfungus which is capable of being propagated in vitro in filamentous formwith a vector comprising DNA coding for a signal peptide and aheterologous polypeptide.
 2. The process of claim 1 wherein said vectorfurther comprises promoter DNA operably linked to said coding DNA. 3.The process of claim 2 wherein such filamentous fungus is from thesubdivision Eumycotina and is capable of being propagated in vitro infilamentous form.
 4. The process of claim 3 further comprising culturingsaid transformed filamentous fungus under conditions which permit theexpression of said coding DNA and secretion of said heterologouspolypeptide.
 5. The process of claim 3 further comprising the step ofisolating said secreted heterologous polypeptide.
 6. A processcomprising culturing a filamentous fungus transformed with a vectorcomprising DNA coding for a signal peptide and a heterologouspolypeptide wherein said filamentous fungus is capable of beingpropagated in vitro in filamentous form.
 7. The process of claim 6wherein said vector further comprises promoter DNA operably linked tosaid coding DNA.
 8. The process of claim 7 wherein such filamentousfungus is from the subdivision Eumycotina.
 9. The process of claim 7wherein said culturing is under conditions which permit the expressionof said coding DNA and secretion of said heterologous polypeptide.
 10. Afilamentous fungus transformed with a vector comprising DNA coding for asignal peptide and a heterologous polypeptide, wherein said filamentousfungus is capable of being propagated in vitro in filamentous form. 11.The filamentous fungus of claim 10 wherein said vector further comprisespromoter DNA operably linked to said coding DNA.
 12. The filamentousfungus of claim 11 wherein said filamentous fungi is from thesubdivision Eumycotina.
 13. The filamentous fungus of claim 12 whereinsaid filamentous fungus from the subdivision Eumycotina is capable ofbeing propagated in vitro in filamentous form.
 14. A DNA construct foruse in transforming a filamentous fungus to enable secretion of aheterologous polypeptide, said DNA construct comprising promoter DNAoperably linked to coding DNA, said coding DNA comprising DNA coding fora signal peptide and said heterologous polypeptide.